It is a universal and long-term desire that foods should comply with individual health, religious rituals, budget and choice. Multiple targets detection in a single assay platform is a technological challenge but has potential to significantly cut analysis cost and time, offering easy analysis of food components in the industry and retail outlets. The multiplex PCR systems developed here conveniently detected five meat species forbidden in Islamic foods with 0.01–0.02 ng sensitivity, clearly showing its appeal in halal food industry and halal regulatory bodies. Instead of single species, the detection of five different species in a single assay platform would clearly cut the analysis cost by at least five times. Shorter amplicon lengths (108–172 bp) and successful amplification of target species under raw and extremely heat treated meatballs suggested that the method could be used in the screening of target species under compromised states and complex matrices.